This proposal outlines the development of a new product, and a new methodological approach to the simultaneous measurement of subdermal PO2 and hemoglobin oxygen saturation in skin. The product, Oxyspheres, will find wide use in the development of healing therapies for skin grafts, skin ulcers, and amputations. The capability to measure PO2 at the interface between skin and underlying tissues and to measure hemoglobin saturation in the skin with a single product will assist researchers and clinicians in evaluating the effects of oxygen on healing and the effects of therapies on altering local oxygen availability. The luminescence emission spectra of Oxyspheres at different oxygen concentrations will be curve fit using optimization methods and a multisite model. Typically, multiwavelength intensity studies of subsurface luminescence are complicated by wavelength-dependent attenuation of light by absorbing components (oxy- and deoxy-hemoglobin and melanin) and by tissue light scattering. The methodological approach for using Oxyspheres will take advantage of the known spectral behaviors of these components to fit Oxyspheres luminescence emission spectra and to determine PO2 and hemoglobin oxygen saturation from the fitting parameters. This innovative approach represents a significant advancement in measuring oxygen availability in wounds, and it also makes a critical advancement in using fluorescent probes to measure tissue biochemistry and physiology in vivo. The new methods will allow multiwavelength analysis of fluorescence emission to quantify concentrations of analytes in overlying tissues while compensating for wavelength dependent attenuation of emission intensities. Development of the method will follow a stepwise development using samples matrixes of increasing complexity, from buffer to hemoglobin solutions, to skin grafts. Phase I research has well defined milestones and feasibility criteria for each of the three stages of development. Phase II will advance the products and analytical methods into commercial products to be used in clinical practice. PUBLIC HEALTH RELEVANCE: This research will develop an innovative new product that can measure tissue oxygen tensions and hemoglobin oxygen saturations simultaneously. The ability to measure oxygen available at specific locations in tissue in addition to measuring oxygen available from the circulation provides a powerful new tool to physicians developing methods to enhance skin healing in patients suffering from diabetes, pressure ulcers, skin grafts, and amputations. The methodological approach applied to these problems promises to have general application to many fluorescent probes of cellular biochemistry and physiology and to become a generally applied method for analyzing tissue behavior in living systems.